CN101287991A

CN101287991A - A method for quantification of allergens

Info

Publication number: CN101287991A
Application number: CNA2006800383055A
Authority: CN
Inventors: U·塞帕拉
Original assignee: ALK Abello AS
Current assignee: ALK Abello AS
Priority date: 2005-09-15
Filing date: 2006-09-01
Publication date: 2008-10-15
Anticipated expiration: 2026-09-01
Also published as: PT1931998E; CN101287991B; DK2228656T3; ES2347277T3; DK1931998T3

Abstract

The present invention relates to method for quantification of the absolute amount of allergen in an allergen sample comprising : a) providing a known amount of one or more allergen calibration standard peptide(s) having a sequence of amino acids which is identical with, and optionally unique for, a sequence to be found in the allergen to be quantified and optionally labelling said allergen calibration standard peptide(s), b) degrading the allergen sample to obtain a mixture of peptides, and optionally labelling said peptides with one or more labelling agent(s), wherein at least the peptides in the degraded allergen sample or the calibration standard peptides are labelled, and if both the peptides in the degraded allergen sample and the allergen calibration standard peptide(s) are labelled, the labelling agent(s) used for labelling the allergen calibration standard peptide(s) are different from the labelling agent(s) used for labelling the peptides of the degraded allergen sample, c) quantifying the absolute amount of allergen by correlating the amount of the allergen calibration standard peptide(s) with the amount of the corresponding peptide(s) of the degraded allergen sample by mass analysis.

Description

Allergen is carried out quantitative methods

Technical field

The present invention relates to allergen is carried out quantitative field.

Background of invention

Allergen is to induce human abnormalism to reply the antigenicity molecule that produces with IgE antibody.They can be used for diagnosis and treatment allergic reaction, that is, the allergen immunotherapy, the latter is the allergen vaccine form.Human placed oneself in the midst of cause the allergia source material, for example food, pollen or acarid faecal particles are natural just exists as the complex mixture of main and minor allergen.Main allergen is that this source is produced allergic Most patients to its allergen that reacts to some extent.But, seem that any albumen all is possible allergen, because, identified increasing minor allergen along with cognition increases.

Because the complicacy in allergen source is at first inferred some allergenic amino acid sequences from the nucleotide sequence that is derived from cDNA.Clone to the allergenic gene of encoding discloses, and most of allergens are heterogeneous, and they are as the potpourri existence of different allergen and variant.Homology allergen and different allergenic amino acid sequence comparison are shown, can identify them by constant and Variable Area sequence, and/or they can be divided into constant and the Variable Area sequence.The constant region domain amino acid sequence is unique for kind, but the Variable Area amino acid sequence all is unique concerning every kind of mutation should be former then.

By using standardized natural allergenic extract (it further is formulated as allergen vaccine), carry out traditional allergen specific immunotherapy and diagnosis at present.These water-based vaccines are for example set and careless pollen, dust acarid culture and animal hair and scurf particle based on causing allergic natural origin material.The composition of these natural source materials has been notified variation considerably, and this variation depends on that collection causes the when and where of allergia source material.In addition, also can use a plurality of kinds, commercial allergen vaccine is formulated as allergenic potpourri.

Cognition to the composition of important allergen inclusions and extract is the condition precedent of final product repeatability, safety and effect.The a major challenge of producing allergen vaccine is standardization,, guarantees that different batches has constant effectiveness that is.In view of starting material are natural origins, change quite bigly, therefore need control by means based on science.The composition of extract should reflect the composition of the water soluble component that causes the allergia source material ideally, because it extracts at respiratory tract (airways) mucomembranous surface, and is and passs human immune system.But all extracts all contain some allergens, and they cause total IgE combination at each patient with various combination.Therefore, ideally, need be controlled all components, but for present technology, this also can't realize from quality and quantity two aspects.

Carry out standardization with a variety of distinct methods at present, because all there is the exclusive standardization flow process of company in manufacturer of every family.By technology such as SDS-PAGE, isoelectric focusings, and panimmunity electrophoresis (QIE) and elisa technique (using monoclonal antibody and/or polyclonal antibody) and radiation allergen absorption (RAST) or correlation technique are carried out standardization.A kind of different batches standardization of optimum, for example SQ standardization flow process has the flow process of three steps basically: 1) guarantee optimum composition and the constant ratio between all components by the sxemiquantitative immuno-electrophoresis, 2) measure main allergen component by quantitative immunoelectrophoresis, and 3) as at Magic

That measures in the check is such, regulates total IgE in conjunction with effectiveness.In Europe, the distinctive reference preparation of all relative intra-company of all standardization at present carries out, and in the U.S., FDA has issued all manufacturer's accepted standards.All quantitative aspects of these at present used technology all depend on the antibody as reagent, and therefore very fragile, can change along with the time.

Absolute quantitation to specific vaccine component in the allergenic complex mixture is also remarkable, and it also is not set up as sensitive, conventional high-throughput techniques.

In food industry, be used for the food allergen is carried out reliable detection and quantitative conventional high-throughput techniques also is necessary.Because the unexpected cross pollution of production period may cause nut to become the hidden parts of food.Production for example has and do not have, and the company of the similar food product of nut may have difficulty aspect the cleaner production equipment between the dissimilar food of manufacturing.Trace in the food of Sheng Chaning before, for example nut may reside on the equipment.There is not the food of nut then may contain the nut of trace via first of same device fabrication.Because the cross pollution of nut or peanut causes allergic food to be, for example, chocolate, candy, biscuit, dessert, sugar, fried circle (donuts), cereal, milk shake, oat bar, assorted breakfast wheat, group, steamed sponge cake, ice cream, barbecue sauce.Milk may cause from dairy products, from milk, based on the formula food of milk or contain allergic effect reaction from a small amount of milk proem of the baby food of the albumen of milk.For fear of producing the pollution that milk proem causes milk allergic effect type child during baby food or the infant formula, therefore need be at allergenic detection of milk and quantivative approach.For guaranteeing to meet the food labelling regulation and, being essential at allergenic reliable detection of food and quantivative approach in order to promote protection to the consumer.Physico-chemical process has been described, for example, mass spectrum, and immunological method.Common sensitivity, specificity, repeatability, precision and accuracy standard must satisfy.But the problem that still has cross reactivity, matrix effect (matrixeffects) and food processing aspect at present.Its biologic activity may still keep when albuminous degeneration.

Biological mass spectrometry (MS) at first is used to the molecular weight and the characteristic of evaluating protein matter and peptide.In the last few years, mass spectral development made and can be used for occurring carry out quantitative technology from the various biomolecules of complex mixture (for example, blood plasma, cell and tissue sample).Early stage quantitative technique is only set up the relative quantification of protein, and more recent technology then can be assessed the absolute magnitude of molecule (s) of interest.Developing rapidly of quantitative technique mainly is because the progress in proteomics field, particularly to for example healthy and morbid state is distinguished and to the progress of the application identified at the marker molecule of some kinds of diseases (for example, cancer, rheumatic arthritis and Alzheimers disease).The major advantage of these quantitative techniques of being undertaken by MS is the high sensitivity of technology, and it is in the scope of 300amol to 300fmol sample.

WO 2004/070352 discloses a kind of method, is used to use the group with amount dystopy (isobaric) labelled reagent or isobar labelled reagent, with respect to internal standard peptide is carried out quantitatively.

US 6,872, and 575 disclose a kind of method, be used for one or more protein of complex sample potpourri are identified, and need not this albumen of purifying or obtain its compound peptide mark (composite peptide signature).

US 6,864, and 089 discloses a kind of method, are used for by peptide or protein sample are carried out the difference isotope labeling quantitatively.

Be used to use the MS technology that albumen is carried out other quantitative method to be, for example, the AQUA technology, it uses the inner peptide of proofreading and correct, this be with the stable isotope that mixes ( ¹³C, ¹⁵N) synthesize, trypsase carries out native peptides (the Stemmann Oet al.Cell 2001 that enzymatic digestion forms by for example using with simulation; 107 (6): 715-26, Gerber SA et al.Proc Natl AcadSci USA 2003; 100 (12): 6940-5).

Other method is ICPL (isotope-coded protein labeling) method, and as Kellermannet al, Proteomics 5, and 4-15 is described, wherein for example uses ¹²C/ ¹³C ₆-niacin-succinimide is as the ICPL mark.

Mass spectrum at first is introduced into the allergic reaction research field, with the characterization natural allergen, comprises posttranslational modification, for example, and the glycosylation situation.Also further characterize different allergen of reorganization and/or variant with it, wherein much express in multiple expression system, described expression system is Esherica Coli, Pichia Pastoris and Baculovirus expression system for example.

Johannes et al, J Allergy Clin Immunol, Vol.110, the 131-138 page or leaf of No.1 (2002) has been described the actual expression of studying the allergen isotype of identifying out by the PCR clone (isoform) with MS, at Swoboda et al., J.Biol Chem, Vol 270, in the 2607-2613 page or leaf of No.6 (1995), clone the isotype of analyzing main birch pollen allergen Bet v1 with liquid chromatography, MS and cDNA.

But at present the method for following sensitivity still has demand, and described method is such: can be in addition quantitative by it to the active component in the vaccine for example, described active component for example, from same kind or different types of allergen and/or different allergen.Use the method for the amino acid sequence of MS technology and kind specificity and allergen specific that a kind of very sensitive method is provided, by this method, can be in addition quantitative to the content of the group of specific allergen or allergen (different allergen or homology allergen).The method according to this invention can be used for, for example, in discharge detecting, with during guaranteeing production of vaccine, in the final products and to allergenic amount safety in a plurality of stages of the storage of active component and/or product, accurately.This method also will be beneficial to exploitation second generation allergen vaccine, for example, wherein use the vaccine of recombinant allergen as active component.These class methods make and can optimize active component in the second generation allergen vaccine based on the composition of present vaccine and/or to its cognition.

Summary of the invention

According to the present invention, a kind of method is provided, be used for the allergen from multiple source is carried out absolute quantitation.

According to an aspect, the invention provides a kind of method, be used for the allergenic absolute magnitude of allergen sample quantitatively in addition, described method comprises the steps:

A) provide one or more allergen calibration standard peptides of known quantity, its have with treat quantitative allergen in the identical amino acid sequence of sequence found, and alternatively, to described allergen calibration standard peptide mark in addition,

B) degraded allergen sample, obtaining the potpourri of peptide, and alternatively, with one or more labelled reagents to described peptide mark in addition,

Wherein, have at least equally in peptide in the allergen sample of degraded or the calibration standard peptide and be labeled, if the two all has been labeled peptide in the allergen sample of degraded and allergen calibration standard peptide, the labelled reagent that is used for mark allergen calibration standard peptide is different with the labelled reagent of the peptide of the allergen sample that is used for the mark degraded

C) by quality analysis, the amount of allergen calibration standard peptide and amount from the corresponding peptide in the allergen sample of degraded are associated, come allergenic absolute magnitude in addition quantitative.

In one embodiment of the invention, treat quantitative allergen by

More than a kind of different allergen, for example constitute from member same kind, that have the allergen group that surpasses 67% amino acid sequence identity.

In another embodiment of the present invention, treat that quantitative allergen is by constituting more than a kind of homology allergen.

According on the other hand, the invention provides a kind of method, be used for the allergenic absolute magnitude of allergen sample quantitatively in addition, described method comprises the steps:

A) provide one or more allergen calibration standard peptides of known quantity, it has unique amino acid sequence the sequence of finding in treating quantitative allergen or different allergen, and, alternatively, to described allergen calibration standard peptide mark in addition,

Wherein, have at least equally in peptide in the degraded sample or the calibration standard peptide and be labeled, if the two all has been labeled peptide in the degraded sample and allergen calibration standard peptide, the labelled reagent that is used for mark allergen calibration standard peptide is different with the labelled reagent of the peptide that is used for mark degraded sample

In one aspect of the invention, provide with treat quantitative different allergen or homology allergen in the identical amino acid sequence of the sequence found as the purposes of allergen calibration standard peptide, be used for allergen is carried out absolute quantitation, and alternatively, allergen identified.Preferably, the degraded in the step b) causes producing peptide mixer, wherein, and a kind of the comprising and the identical amino acid sequence of allergen calibration standard peptide in the peptide.

In another aspect of this invention, provide following amino acid sequence to be used for allergen is carried out absolute quantitation and alternatively as allergen calibration standard peptide, to the purposes that allergen is identified, wherein said amino acid sequence be for treat quantitative allergen or mutation should be former unique sequence.Preferably, the degraded in the step b) causes producing peptide mixer, and wherein, a kind of in the peptide comprises the amino acid sequence identical with the allergen calibration standard.

In still another aspect of the invention, provide a kind of method, be used to obtain allergen calibration standard peptide, quantitative in addition to be used for different allergen or homology allergen, wherein, described allergen calibration standard peptide obtains by following method:

By different allergen or the allergenic sequence of homology are compared, identify constant amino acid sequence in treating quantitative different allergen or homology allergen, and preparation has the synthetic property allergen calibration standard peptide of this constant series.

Also aspect one, provide a kind of method of the present invention, be used to obtain allergen calibration standard peptide, in addition quantitative to be used for allergen or different allergen, wherein, described allergen calibration standard peptide obtains by following method:

By allergen or different allergenic sequence are compared with other different allergen and/or homology allergen, identify for treat quantitative allergen or mutation should be former unique variable amino acid sequence, and the synthetic property allergen calibration standard peptide of preparation with this unique sequences.

The accompanying drawing summary

Fig. 1. by carrier NTI software (Invitrogen), (Fig. 1 is a) and Bet v1 (Fig. 1 b)---the amino acid sequence that main birch allergen carries out is compared to acarid group 2 allergen kinds.Being assessed as the possible amino acid sequence runic that can be used as inner calibration standard peptide (can be used for should be former quantitative to mutation) emphasizes to illustrate.

Fig. 2. by trypsase to house dust acarid allergen: a) Der f2 and b) Derp2, c) Phl p1, d) Phlp 5a, e) Phl p 5b and f) the theoretical enzymatic cutting carried out of Bet v1 (GPMAW, Lighthouse data).Select for use the kind specific peptide that carries out detection by quantitative to emphasize to illustrate with runic and grey.

Fig. 3 .a) to MALDI-TOF MS fingerprint analysis purified and that carry out through the potpourri of the natural Der f2 of trypsinization and Der p2 (1: 1), and b) to MALDI-TOF MS fingerprint analysis purified and that carry out through the potpourri of the reorganization Der f2 of trypsinization and Der p2 (1: 1).

Fig. 4. the SDS-PAGE that is undertaken by HDM (the house dust acarid) allergenic extract that uses hydrophobic interaction chromatograph to separate is analyzed.The fraction of HDM albumen is divided into two large protein storehouses (I and II), further carries out quantitative test again.

Fig. 5. utilizing ITRAQ to allergenic in quantitatively ^TM(CA in USA) when chemistry, carries out the strategy of mark to sample for Applied Biosystems, Foster City.

Fig. 6. to a) peptide 1 through the iTRAQ mark, m/z2353.44 (Der p2,32-48), b) peptide 2, m/z 2326.35 (Der f2,32-48), c) is through trypsinization and through the nDer of iTRAQ mark p2 peptide and d) MS that carries out of nDer f2 peptide analyzes.

Fig. 7. the MS/MS fragmentation that the potpourri of nDer f2, nDer p2, peptide 1 and peptide 2 is carried out.The amount of different allergen nDer p2 (114.10) and Der f2 (115.10) is calculated as in the sample mixture: the ratio (peptide 2) of the ratio (peptide 1) of the signal area of m/z 114 and the area of m/z 116 and the area of m/z 115 and the area of m/z 117.

Fig. 8. the anti-phase analysis that the potpourri from nDer f2, nDer p2, peptide 1 and the peptide 2 of SCX chromatogram is carried out.Analyze the peak of putting on the identification of M ALDI-TOF target with MS and MS/MS.

Fig. 9. the MS that the potpourri through the nBet of trypsinization v1 and inner calibration standard (AQUA peptide) is carried out analyzes.The mass discrepancy of 6Da is shown in the last angle of figure between native peptides and the calibration standard.

Detailed Description Of The Invention

In this context, term " allergen " refers to: be reported as to give in its repeated exposure and can induce the allergic effect reaction when individual, that is, the naturally occurring albumen of the reaction of IgE mediation, modified albumen, recombinant protein, recombination mutation body protein or its any protein fragments or protein mixture.

Naturally occurring allergenic example comprise pollen allergen (tree, weeds, herbaceous plant and showy flowers of herbaceous plants powder allergen), acarid allergen (from, for example, house dust acarid and reserve acarid), insect allergen (suction, saliva source with the allergen venom source), animal allergen (such as from saliva, hair and scurf such as dog, cat, horse, rat, mouse etc.), fungi allergen and food allergen.

From tree, careless and herbal important pollen allergen be following these, it is from the Balanopsidales on the taxology (Fagales), sweet-scented osmanthus order (Oleales), pine China fir order (Pinales) and plane tree order (platanaceae), comprise birch (Betula), alder (Alnus), hazel (Corylus), hornbeam (Carpinus), Chinese olive tree (Olea), cdear (Cryptomeria and Juniperus), plane tree (Platanus), Poales (Poales), comprise, i.a. Lolium (Lolium), ladder forage spp (Phleum), Poa L. (Poa), Cynodon (Cynodon), orchardgrass (Dactylis), Holcus (Holcus), phalaris arundinacea (Phalaris), the grass of Secale (Secale) and sorghum (Sorghum), and chrysanthemum order (Asterales) and Urticales (Urticales), comprise i.a. Ambrosia (Ambrosia), the herbaceous plant of artemisia (Artemisia) and Parietaria (Parietaria). Other important suction allergen is from Dermatophagoides (Dermatophagoides) and has a liking for the house dust acarid that mould mite belongs to (Euroglyphus); the reserve acarid; for example; Lepidoglyphys, Glyciphagus (Glycyphagus) and Tyrophagus (Tyrophagus); from cockroach, mosquito and flea; for example; those of Blatella (Blatella), Periplaneta (Periplaneta), Chironomous (Chironomus) and Ctenocepphalides; from mammal; those of cat, dog and horse for example; the venom allergen; comprise following these; it is from the insect that stings or sting; for example; from Hymenoptera on the taxology (Hymenoptera), comprise those of honeybee (honeybee (Apidae) Superfamily), wasp (Vespidea Superfamily) and ant (ant (Formicoidae) Superfamily). Important suction allergen from fungi is, i.a. is from those of Alternaria (Alternaria), Cladosporium (Cladosporium), aspergillus (Aspergillus) and Penicillium (Penicillium).

The example of food allergen from wheat (for example is, Tri a 18-19), shell-fish food, comprise, shrimp (for example, Met e1, Pen a1, Pen I1, Pen m1 and Pen m2), prawn, crab and lobster, fish (for example, Gad c1 and Sal s1), hen egg (Gal d1 for example, Gal d2), peanut (for example Ara h1-8), soybean (Gly m 1-4), milk (Bos d 4-8), nut, for example, almond (Pru du 4), Brazil nut (Ber e1, Ber e2), cashew nut (Ana o 1-3), (for example, Cor a 1.04 for fibert, Cor a2, Cor a8) and English walnut (Jug n 1-2 for example, Jug r 1-3), celery (Api g1, Api g4, Api g5), mustard (Sin a1 and Bra j1) and sesame seed (Ses i 1-6), particularly from wheat (for example Tri a 18-19), hen egg (for example, Gal d1, Gal d2), peanut (for example, Ara h 1-8), soybean (Gly m 1-4), the allergen of milk (Bos d 4-8).

The example of recombinant allergen includes but not limited to, use the recombinant technique preparation, from the albumen/peptide of plant pollen, showy flowers of herbaceous plants powder, tree pollen, weeds pollen, insect venom, dust and reserve acarid albumen, animal scurf, saliva, fungal spore and food allergen (that is, peanut, milk, wheat bran and egg). Recombinant allergen can be for example with extensive acquisition; This realizes by the microbial expression system that is incubated at cocoa on the fermentation tank; Wherein said microflora produces by recombinant DNA technology; Perhaps; When synthetic by chemical method; by obtaining with precursor or other chemicals. ， rBet v1、rAln g1、rCor a1、rCar b1、rCry j1、rCry j2、 rOle e1、rAmb a1、rArt v1、rCyn d1、rDac g1、rLol p1、 rLol p5、rPhl p1、rPhl p5、rPoa p1、rPoa p5、rSor h1、 rDer f1、rDer f2、rDer p1、rDer p2、rEur m1、rEur m2、 rGly d1、rLep d2、rBla g1、rBla g2、rFel d1、rCan f1、 rCan f2、rBos d2、rEqu c1、rEqu c2、rMus m1、rApis m1、 rApi m2、rVes v1、rVes v2、rVes v5、rDol m1、rDol m2、 rDol m5、rPol a1、rPol a2、rPol a5、rAlt a1rCla h1 （ r ）。

Recombinant mutant allergen and wild type are different, because by genetic manipulation method allergenic gene is modified, so that the polypeptide of their codings shows independent or several amino acid whose replacements, disappearance and/or interpolation than wild type. The allergenic example of recombinant mutant comprises that allergen replaces variant, adds variant, oligomer, fragment, disappearance variant, hybrid molecule and other variant.

Modified allergenic example comprises such allergen, and its naturally occurring form is relevant with allergic disease situation among the responsive experimenter, wherein, described modified recombinant allergen than naturally occurring allergen through changing. The present invention includes the allergen variants that contains the exchange of several amino acid, Allergen variants, oligomer, fragment, disappearance variant, hybrid molecule, myristyl, glycosylated, palmitoylation and allergen phosphorylation and other variant. Modified allergen can produce for example mixing of site-directed mutagenesis method, PCR method, chemical synthesis and these methods by any method that is fit to.

In one embodiment of the invention, treat that quantitative allergen is selected from one or more in following group: Bet v1, Aln g1, Cor a1 and Car b1, Que a1, Cry j 1, Cry j2, Cup a1, Cup s1, Jun a1, Jun a2, Jun a3, Ole e1, Lig v1, Syr v1, Pla 11, Pla a1, Pla a2, Amb a1, Amb a2, Amb t5, Art v1, Art v2, Art v3, Par j1, Par j 2, Par j3, Sal k1, Ave e1, Cyn d1, Cyn d7, Dac g1, Fes p1, Hol 11, Lol p1 and 5, Pha a1, Pas n1, Phl p1, Phl p2, Phl p3, Phl p4, Phl p5, Phl p6, Poa p1, Poa p5, Sec c1, Sec c5, Sor h1, Der f1, Der f2, Der f3, Der f7, Der p1, Der p2, Der p3, Der p7, Der m1, Eur m1, Eur m 2, Gly d1, Gly d2, Lep d1, Lep d2, Blo t1, Tyr p2, Bla g1, Bla g2, Per a1, Per a3, Per a7, Fel d1, Fel d2, Fel d3, Fel d4, Can f1, Can f2, Bos d2, Equ c1, Equ c 2, Equ c3, Mus m1, Rat n1, Apis m1, Api m1, Api m2, Ves v1, Ves v2, Ves v5, Ves f5, Ves g5, Ves m1, Ves m2, Ves m5, Ves p5, Ves s5, Ves vi 5, Dol m1, Dol m2, Dol m 5, Dol a5, Pol a1, Pol a2, Pol a5, Sol i1, Sol i2, Sol i3 and Sol i4, Alt a1, Alt a3, Alt a4, Alt a5, Alt a6, Cla h1, Cla h2, Cla h6 Asp f1, Bos d4, Mal d1, Mal d3, Gly m1, Gly m2, Gly m3, Ara h1, Ara h2, Ara h3, Ara h4, Ara h5 or its be heterozygote arbitrarily.

In another embodiment of the present invention, treat that quantitative allergen is selected from one or more in following group: showy flowers of herbaceous plants powder allergen, Phl p1 for example, Phl p5, Phl p6, Poa p1, Poa p5, Dac g1, Fes p1, Lol p1 and Lol p5, dust acarid allergen, for example, Der f1, Der f2, Der p1 and Der p2, the venom allergen, Api m1 for example, Api m2, Ves v1, Ves v2, Ves v5, Dol m1, Dol m2, Dol m5, Dol a5, Pol a1, Pol a2 and Pol a5, the weeds allergen, Amb a1 for example, Amb a2, Par j1, Par o1 and Par m1, the birch allergen, Bet v1 for example, the Japanese cedar allergen, for example Cry j1 and Cry j2, cockroach allergens, Per a1 for example, the Chinese olive tree pollen allergen, Ole e1 for example, the cat allergen, Fel d1 for example, the dog allergen, for example Can f1 and Can f2, horse allergen, for example Equ c1 and Equ c2, mugworth allergen, for example Art v1, Art v2, Art v3, the mould allergen, Alt a1 for example, Alt a3, Alt a4, Alt a5, Alt a6, Cla h1, Cla h2 and Cla h6 and fiery ant allergen, for example Sol i2, Sol i3 and Sol i4.

In another embodiment of the invention, treat that quantitative allergen is selected from one or more in following group: showy flowers of herbaceous plants powder allergen, for example Phl p1, Phl p5 and Phl p6, the Chinese olive tree pollen allergen, Ole e1 for example, dust acarid allergen, for example Der f1, Der f2, Der p1 and Der p2, the venom allergen, for example Ves v1, Ves v2 and Ves v5, weeds allergen, for example Amb a1, Amb a2, Par j1, Par o1 and Par m1 and tree allergen, for example Bet v1, Cry j1 and Cry j2.

In another embodiment of the invention, treat that quantitative allergen is to be selected from the different allergen of Der f1, Der p1, Der f2 and Der p2 one or more.

In an also embodiment of the present invention, treat that quantitative allergen is to be selected among Phl p1, Phl p5, Phl p6, Poa p1, Poa p5, Dac g1, Fes p1, Lol p1, the Lol p5 one or more.

In an again embodiment of the present invention, treat that quantitative allergen is to be selected from the different allergen of Amb a1 and Amb a2 one or more.

Can consisting of by several closely similar molecule from the allergen of single kind. When these similar molecules are shared following common biochemical property, just be named as different allergen, described character is: the molecular size that a. is similar; If b. know, identical biological function, for example, the enzyme effect; And the amino acid sequence identity of c.＞67%. In this context, have＞67% amino acid sequence identity and be called as different allergen from the allergen group membership of one species. Every kind of different allergen can have the closely similar sequence of various ways, and described sequence only has a small amount of otherness amino acid sequence, and these are called as variant, and in the scope of the term " different allergen " in this context of also falling.

In this context, term " homology allergen " (for example refers to be considered to share similar three-dimensional structure, molecular size, identical biological function (if knowing), the enzyme effect), from different types of allergen, they can share the structure epi-position for IgE antibody. In another embodiment of the invention, the homology allergen has＞20% amino acid sequence identity, shares common constant amino acid sequence, preferably, and 2-20 amino acid whose sequence at least, more preferably, 4-15, most preferably, 6-10.

For example, Amp m2 and Ves v2 and Def f2 and Der p2 can be used as the allergenic example of homology.

In this context, " allergenic extract " this expression refers to by extracting any extract of the original raw material acquisition of biological allergic effect, such as " Allergenic extracts ", H. Ipsen et al, chapter 20 in Allergy, principle and practice (Ed. S.Manning) 1993, Mosby-Year Book, the generality of St.Louis is described described. This type of extract can obtain like this: water-soluble material carried out water-based extracts, then carry out purification step (for example filtering), and to obtain solution, that is, and extract. Then can carry out further purifying and/or processing to extract, for example freeze drying is basically to remove whole water. Usually, allergenic extract comprises the mixture of protein and other molecule. Allergen protein is classified as main allergen, intermediate product allergen, minor allergen or usually without classification. Allergenic extract comprises main allergen and minor allergen usually. Main allergen will consist of about 5-15% of average allergenic extract usually, be more typically about 10%. Based on the assessment to special allergen clinical importance, it provides hereinafter to allergenic classification. The important main allergen of finding in extract comprises:

grass group

1 and 5 and 6 allergens (for example Phl p1,5 and 6),

dust acarid group

1 and 2 allergens (for example Der p1, Der p2), tree pollen allergen 1 (Bet v1),

cdear pollen allergen

1 and 2 are (for example, Cry j1, Cry j2), Ambrosia pollen 1 and 2 (Amb a1, Amb a2), cat allergen 1 (that is, Fel d1).

" biology allergen source material " this expression that is used for herein refers to comprise one or more allergenic any biologic materials. The example of materials is mite PMB (pure acarid polypide, Pure Mite Body) or WMC (total acarid culture, Whole Mite Culture), degreasing or non-degreasing pollen from for example grass, herbaceous plant, weeds and tree, animal hair and scurf, fur, fungal mycelium and spore, the insect polypide, venom or saliva and food.

Biology allergen source material can comprise the contaminative material, for example foreign pollen and from the remains of the plant of allergen pollen source material and flower. The maximum contamination level of acceptable pollen from other kind is 1%. It also should not contain flower and the remains of plant, is 5% to its weight limits.

The term that uses in this paper context " allergen vaccine " comprises at least a allergen, it can be originated from same allergic effect source or from the different allergia that causes, for example, organize 5 allergens or acarid group 1 and group 2 allergens from grass group 1 and the grass of different acarids and grass seeds class respectively, the weeds allergen, for example lack and large artemisiifolia allergen, different fungi allergens, for example Alternaria and Cladosporium, the tree allergen, for example birch, hazel, hornbeam, Oak Tree and alder allergen, food allergen, for example peanut, soybean and milk allergen.

The vaccine production method is normally well known in the art.Typically, vaccine is prepared as injectable, for example as liquid solution or suspending liquid.This type of vaccine also can be emulsified or preparation, so that can nasal administration and oral, comprises through cheek and sublingual administration.Target immunogenicity component can suitably be mixed with excipient, and excipient is pharmaceutically useful, and compatible with active component.The example of suitable excipient is water, salt solution, dextrose, glycerine etc. and combination thereof.Vaccine can additionally contain other material, for example the adjuvant of wetting agent, emulsifying agent, buffering agent or enhancing vaccine validity.

According to an aspect of the present invention, provide a kind of method, be used for the allergenic absolute magnitude of allergen sample quantitatively in addition, described method comprises the steps:

In a kind of special embodiment of the present invention, the peptide of calibration standard peptide and degradation samples all is labeled, but comes mark with different labelled reagents.

In another embodiment of the invention, the calibration standard peptide is labeled, but the peptide of degradation samples is not labeled.

According to another embodiment of the present invention, allergen calibration standard peptide is not labeled, but the peptide of the allergen sample of degraded has been labeled.

Mentioned above of the present invention a kind of preferred embodiment in, a kind of allergen calibration standard peptide is provided in step a).Therefore, preferably,, only use a kind of allergen calibration standard peptide at every kind of allergen sample.

According to of the present invention another preferred embodiment, when the degradation samples in the step b) is labeled, only use a kind of labelled reagent to its mark in addition.

Therefore, preferably, only provide a kind of allergen calibration standard peptide in step a), the degradation samples in the step b) is also only by a kind of labelled reagent institute mark.

In addition, when the calibration standard peptide was labeled, it was preferably only by a kind of labelled reagent institute mark.

Quality analysis, for example MS etc. can carry out on the potpourri special allergen sample, according to allergen calibration standard peptide provided by the invention to the allergen sample with at this some.

In this paper context, term " allergen calibration standard peptide; its have with treat quantitative allergen in the identical amino acid sequence of sequence found " the such amino acid sequence region of expression, it is in treating quantitative allergenic different allergen group or treating that in the quantitative homology allergen group be constant, that is, identical.According to of the present invention preferred embodiment a kind of, allergen calibration standard peptide is selected to be, make and treat that the degraded of quantitative allergen (different allergen or homology allergen) in step b) causes producing peptide mixer, wherein, a kind of the comprising and the identical amino acid sequence of allergen calibration standard peptide in the peptide in this potpourri.

Also can be quantitative in addition according to the present invention to specific different allergen or allergen.

According to this aspect, the invention provides a kind of method, be used for specific allergen of allergen sample or different allergenic absolute magnitude quantitatively in addition, described method comprises the steps:

A) provide one or more allergen calibration standard peptides of known quantity, it has for unique amino acid sequence the sequence of finding in treating quantitative allergen or different allergen, and, alternatively, to described allergen calibration standard peptide mark in addition,

In a kind of special embodiment of the present invention, the peptide of calibration standard peptide and degradation samples all is labeled, but is to use different labelled reagents to come mark.

In another embodiment of the present invention, the calibration standard peptide is labeled, and the peptide of degradation samples is not labeled.

According to another embodiment of the invention, allergen calibration standard peptide is not labeled, and the peptide of the allergen sample of degraded has been labeled.

As indicated above of the present invention a kind of preferred embodiment in, a kind of allergen calibration standard peptide is provided in step a).Therefore, preferably,, only use a kind of allergen calibration standard peptide at every kind of allergen sample.

Therefore, preferably, in step a), only provide a kind of allergen calibration standard peptide, and if mark, the degradation samples in the step b) is also only by a kind of labelled reagent institute mark.

In this paper context, term " allergen calibration standard peptide; it has for amino acid sequence unique the sequence of finding in treating quantitative allergen or different allergen " the such amino acid sequence region of expression, it is for treating that quantitative different allergen or allergen are variable, that is, unique.According to of the present invention preferred embodiment a kind of, treat that the degraded of quantitative allergen in step b) causes producing the potpourri of peptide, wherein, a kind of the comprising and the identical amino acid sequence of allergen calibration standard peptide in the described peptide.

Amino acid whose quantity in the allergen calibration standard peptide is preferably in 2-20 amino acid whose scope, more preferably in the scope of 4-15, most preferably in the scope of 6-15.This quantity depend on be found with sample in the optimum restriction enzyme site of amino acid sequence coupling, that is, and when coming cutting sample by enzyme, the sequence of constant or Variable Area.In addition, allergen calibration standard used according to the invention is depended on mark and the quantivative approach that will use, provide observable signal and fragmentation when in MS equipment, analyzing.

In this paper context, " allergen sample " refers to comprise one or more allergenic samples.

In one embodiment of the invention, the allergen sample allergenic extract that comprises allergenic extract, naturally occurring purified allergen, modified allergen, recombinant allergen, recombinant mutant allergen, any allergen fragment, different allergenic potpourri or the allergenic potpourri of homology or its combination and comprise the purified natural or recombinant allergen of (spiking) of artificial interpolation.

The allergen sample can be the form of final product, the allergen vaccine of tablet or solution form for example, perhaps can be the product/intermediate product that takes out at production period, for example, the product/intermediate product after biological allergen source material or starting material are extracted.

Of the present invention a kind of preferred embodiment in, the allergen sample is the form of the final product or the intermediate product of allergenic extract, tablet form.

In one aspect of the invention, allergenic extract is provided, wherein, allergenic extract comprises natural allergen and recombinant allergen, it is by such acquisition: quantitative in addition to allergenic amount in the natural extract, and in this extract, add recombinant allergen or natural purified allergen, in final extract (for example, manually having added the natural extract of purified natural or recombinant allergen), to obtain the allergen of requirement.

The method according to this invention make in the allergen sample from one or more kinds, exist and one or more allergens with common amino acid sequence carry out while or the disposable possibility that quantitatively becomes as different allergen or homology allergen.

Can use the method according to this invention, simultaneously or in flow process to the allergen sample in the multiple different allergen of kind carry out quantitatively.

Depend on sample, may must use denaturant and buffering solution, to obtain suitable solution.

Contain at sample under the situation of the material that might disturb method of the present invention, specimen preparation can comprise multiple processing, for example uses acetone precipitation, described material is mercaptan for example, for example, and DTT or mercaptoethanol, the scaling agent of high concentration and/or denaturant, for example SDS, octyl group B-D-glucopyranoside and

X-100 and/or active protease or primary amine (but not interested allergen).The damping fluid of recommending and may disturb the alternative scaling agent of the method according to this invention and/or denaturant and material are listed in, for example Applied Biosystems Itraq ^TMReagentsAmine-Modifying Labeling Reagents for Multiplexed Relative andAbsolute Protein Quantification Protocol from AppliedBiosys tems, Foster City, CA is among the USA.

The complexity that depends on sample was presorted level to sample and is separated and may be good before degraded, for example, if exist under the situation of disturbing the molecule that interested allergen in the sample is surveyed.Also may need sample and its preparation and/or any adjuvant (for example aluminium hydroxide or calcium phosphate) are separated/wash-out.In order to obtain more uncomplicated potpourri, can use the plurality of color spectral technology to come sample is carried out fractionated, for example hydrophobic interaction chromatograph, ion-exchange chromatography and/or immune affinity chromatographic.

In one embodiment of the invention, the allergen sample separates acquisition from presorting level, for example, comprises one or more different allergenic allergenic extract fractions.

In one embodiment of the invention, the allergen sample is to separate the fraction of step from presorting level, wherein, according to size, solubleness, electric charge and/or ligand specificity sample has been carried out fractionated.In another embodiment of the present invention, by chromatogram, for example presort level and separate by hydrophobic interaction chromatograph, reverse-phase chromatography, ion-exchange chromatography, size exclusion chromatogram or affinity chromatography, for example, undertaken by hydrophobic interaction chromatograph.

Fig. 4 shows by using hydrophobic interaction chromatograph to presort the example that level is separated to containing HDM group 1 with 2 allergenic intermediate products.Contain

HDM group

1 and 2 allergenic fractions respectively and separate, come it is identified by immunoprecipitation by its physicochemical property.Two kinds of fractions all can be used for quantitative examination then.

In one embodiment of the invention, chromatogram is afterwards to the desalination of allergen sample.

In one embodiment of the invention, before degraded, the allergen sample is reduced, and sealed any cysteine residues, for example, undertaken by alkylation.

According to the present invention, by handling the sample of degrading, to obtain the potpourri of peptide with one or more enzymes.Optional usefulness has the enzyme of very predictable degraded situation, and making can be by the peptide of relatively identifying and quantitatively obtaining with allergen calibration standard peptide.Enzyme can be one or more proteinase, for example, and two kinds of proteinase or one or more other enzymes.The example of proteolytic enzyme comprises: trypsase, papain, pepsin, ArgC, LysC, V8 proteinase, AspN, pronase, chymotrypsin and carboxypeptidase C.For example, proteolytic enzyme---trypsase is serine protease, and it is cutting peptide bonds between lysine or arginine and nonspecific amino acid, produces the peptide that comprises amine end (N end) and lysine or arginine carboxyl terminal amino acid (C end) thus.By this way, be predictable from peptide to the cutting of albumen, they are at existence and/or content from existence in the sample of trypsinization and/or their source protein of content indication.In addition, the unhindered amina end of peptide can be good nucleopilic reagent, and it assists the mark to it.Because enzymatic activity is predictable, the sequence of the peptide that produces from the degraded of the albumen of known array also is predictable.Adopt this information, can produce " theory " peptide information.For example in from the computer-aided analysis that the sub-fragment ions of the mass spectrophotometry of actual sample is carried out, to " theory " peptide segment really determining cause this can be used for identifying one or more peptides.

In one embodiment of the invention, degraded allergen sample before mark, this is by realizing with this sample of partially or completely degrading with at least a enzymic digestion sample.In another embodiment of the invention, enzyme is a proteolytic enzyme, for example trypsase, papain, pepsin, ArgC, LysC, V8 proteinase, AspN, pronase, chymotrypsin and carboxypeptidase C or its combination for example is selected from the group of ArgC, LysC and trypsase or its combination.In an embodiment more of the present invention, enzyme is a trypsase.

If necessary, can be before mark, prepare sample by any method in the some kinds of methods through digestion.

Those skilled in the art are apparent: have multiple possible approach to come mark sample and allergen calibration standard peptide, to introduce different quality rhetorical functions (functionalities) with given way, making quantitatively becomes possibility to the allergen peptide.Can be for example according to WO 2004/070352, US 6,864,089, Stemmann O et al.Cell 2001; 107 (6): 715-26 and Gerber SA et al.Proc Natl Acad Sci USA2003; 100 (12): the described mark that carries out of 6940-5, these documents are quoted by this and are incorporated this paper into.

In one embodiment of the invention, mark ITRAQ ^TMChemistry carry out (AppliedBiosystems, Foster City, CA, USA).

According to this embodiment of the present invention, the allergen sample of degraded and/or the mark of calibration standard peptide are passed through isomerism (isomeric) or same group (for example, the iTRAQ that measures the dystopy labelled reagent ^TMReagent (Applied Biosystems, Foster City, CA, USA)) carry out.In these reagent every kind all contain can with reaction active groups (RG) and unique reporter group (RP) of analysans reaction, it produces unique " mark ion " in MS/MS analyzes.Use X and Y key, these two kinds of groups further are connected to together with shank (LK).The mark of allergen sample to degraded produces the analysans that is called as the RP-X-LK-Y-sample thus.By regulating mass spectrometer, make X and Y all the mode of bonding fragment carry out analysis to analysans.The fragmentation of key X discharges reporter group from analysans, can be independent of analysans then reporter group is measured.The fragmentation of Y key discharges the RP-LK combination from analysans.Therefore, based on fragmentation, the existence and/or the content of analysans in the existence of reporter group and/or content and the sample can be associated.

With for example 4iTRAQ ^TMReagent carries out mark and allows that different allergen samples are carried out absolute quantitation simultaneously (the allergen sample is degraded, and every kind of peptide mixer is with different iTRAQ ^TMThe reagent mark).The possibility that different allergen samples are analyzed simultaneously makes and can compare through the calibration standard peptide of peptide, sample and the known quantity of mark, and quantitative and evaluation becomes possibility in a step mode by using MS/MS thus.

When using ITRAQ ^TMAnd/or during other labelled reagent, can carry out according to the flow process of manufacturer the mark of sample, as shown in Figure 5.

With use MS technology protein being carried out another quantitatively relevant labeling method is, for example, and the AQUA technology, the stable isotope that wherein use usefulness is mixed ( ¹³C, ¹⁵N) peptide is proofreaied and correct in synthetic inside, and for example using with simulation, trypsase carries out native peptides (the Stemmann Oet al.Cell 2001 that enzymatic digestion forms; 107 (6): 715-26, Gerber SA et al.Proc Natl AcadSci USA 2003; 100 (12): 6940-5).

In one embodiment of the invention, to dividing other mark, after mark, mixed before quantitatively through the not peptide and the allergen calibration standard peptide of isolabeling.

Can select suitable evaluation mode according to the mark that how carries out allergen and calibration standard peptide.

In one embodiment of the invention, by the peptide identification and analysis allergen peptide and allergen calibration standard peptide through mark are compared, further clear and definite (positively) identifies allergen.

Also available ion-exchange chromatography and reverse-phase chromatography combination come isolated peptides as two-dimentional chromatogram, and if necessary, reduce the amount of any salt and organic compound with it before MS analyzes.

In one embodiment of the invention, use mass spectrum to carry out quantitatively.

In another embodiment of the invention, can use tandem mass spectrum maybe can be selected other mass spectrum with fragmentation to molion, carry out allergen and/or different allergenic evaluation.This especially is suitable for using iTRAQ ^TMReagent carries out under the situation of mark.

Tandem mass spectrum (and on littler degree, single phase mass spectrum) has following ability: it can be selected and fragmentation according to quality electric charge (m/z) the comparison molion of molion, and fragment (son) ionic spectrum that obtains of record then.More specifically, can produce sub-fragment ions spectrum by the ion of selecting for use being applied dissipation energy level (for example, collision causes disassociation (CID)).For example, can select ion, in the quality analysis second time, it be carried out fragmentation and analyze once more from the quality analysis first time through the mark peptide corresponding to specific m/z ratio.The typical equipments that can carry out this type of series connection quality analysis includes but not limited to, magnetic field four fans (magneticfour-sector), series connection flight time, three utmost points, four utmost points, ion trap and four utmost point flight time of hybridization (Q-TOF) mass spectrometer.

The mass spectrometer of the above-mentioned type can use with multiple ionization source, and described ionization source includes but not limited to: electron spray ionisation (ES I) and substance assistant laser desorpted ionized (MALDI).Ionization source can be used for producing and is used for the not electrically charged kind of the quality analysis first time of fixed charge.Extra mass spectroscopy device and fragmentation method comprise the post-source decay in the MALDI-MS equipment and use the high energy CID of MALDI-TOF (flight time)-TOF MS.About the recent summary of tandem mass spectrometer, referring to, R.Aebersold and D.Goodlett, Mass Spectrometryin Proteomics.Chem.Rev.101:269-295 (2001).About discussion to TOF TOF quality analysis technology, also see United States Patent (USP) 6,319,476, it incorporates this paper by reference into.

Based on being that allergen or homology allergen or specific allergen or different allergen are carried out quantitatively, allergen calibration standard peptide (variable or constant series) is selected.

In one embodiment of the invention, can carry out absolute quantitation (allergenic different allergenic absolute magnitude) to allergen.

When using iTRAQ ^TMDuring reagent, be used for the isomerism of mark allergen peptide or the group of same amount dystopy mark or mark, for example, iTRAQ-114, iTRAQ-115, iTRAQ-116 or iTRAQ-117, the allergen calibration standard peptide that comes mark to select for use.In case with respect at the relative quantity of having been measured by the relative quantity of the reporter group of the peptide of isolabeling not at the reporter group of (multiple) calibration standard peptide, just can calculate in the sample mixture all through the absolute magnitude of the peptide of isolabeling (being typically expressed as concentration and/or quantity) not, and (for example calculate allergenic amount thus, when sample is extract, from the different allergenic absolute magnitude of kind).From through ITRAQ ^TMThe sample of mark obtains MS and MS/MS can for example use 4700 Explorer ^TMSoftware carries out.In addition, can use GPS Explorer software to carry out database search, it will cause and can clearly identify the peptide from MS/MS.Then can be based on the allergen calibration standard peptide of known quantity, that is, the ratio of sample and calibration standard peptide carries out quantitatively with the data that obtain.

The method according to this invention can be used for, in for example discharge detecting, and guaranteeing during production of vaccine, and in final product and to allergenic amount safety and measurable in a plurality of stages of composition and/or product and crude extract storage.The method according to this invention can be used for developing second generation allergen vaccine, for example uses recombinant allergen as active component, and this is by based on the composition of present vaccine and/or to its cognition, and the active component of optimizing in the second generation allergen vaccine is realized.Present vaccine normally is used for allergen preparation that arrogant quantitative change should former kind, and method of the present invention also will help to determine kind of a paraspecific allergenic composition from these allergen potpourris.The method according to this invention can be used for cleaning and confirms (wherein the trace allergen being measured), discharges and detect and to the analysis of middle product and final products.

Can be by using protein and/or nucleotide database, and cutting analysis program and/or carry out external quality fingerprinting experiment, the peptide that can be used as the calibration standard peptide made.

In this paper context, term " allergen calibration standard peptide " refer to have with different allergen or homology allergen group in the allergen calibration standard of the variable or amino acid sequence that constant series (depending on that it is used for the allergen that is made of unnecessary a kind of different allergen or homology allergen is carried out quantitatively still specific allergen or different allergen being carried out quantitatively) is identical.Preferably synthesize and prepare allergen correction peptide by peptide.

In some cases, interested allergenic sequence is known.Tabulation can for example (www.allergen.org) finds in the website in allergenic official, and this is safeguarded by I.U.I.SAllergen Nomenclature Sub-committee.Can be from protein and nucleotide database, for example, Uniprot Knowlegdebase obtains known allergen sequence.Can use, for example, Sequence Retrieval System (SRS) or use key word, for example, enter title (ID), description (DE), gene name (GN), kind (OS) and/or the organelle (OG) of submission are searched for protein and/or nucleotide sequence.By using VectorNTI software (Invitrogen) and/or, carrying out to protein of interest matter/allergenic further analysis by using ExPASy (Expert Protein Analysis System) the proteomics server of Swiss Institute ofBioinformatics (SIB).Use Blast to search for the sequence alignment that carries out existing allergen obform body and allergen kind, the Blast search can be used for comparing homologous protein and/or nucleotide sequence.Sequence relatively provides for example, the approach that new sequence and gene that characterized in the past and/or albumen are compared.To homology allergen or different allergenic sequence alignment can be used for confirming in the allergen kind with kind between identical (constant) and variable sequence, as shown in Figure 1.

For obtaining optimum calibration standard peptide, can use cutting (degraded) routine analyzer, simulate at interested allergenic cutting analysis.(Lighthouse data, Odense Denmark) analyze the allergen sequence available GPMAW program, and this program is to make to support what MS analyzed.Can infer the cutting (degraded) of allergen sequence is analyzed that described proteinase is trypsase, Asp-N and Lys-C and/or two or more combination in them for example at some kinds of known protein enzymes.Verify optimum digestive enzyme with the theoretical peptide (Fig. 2) that obtains then, and further design the synthetic peptide that can be used as the calibration standard peptide.

On the other hand, can test from external quality fingerprinting and infer the calibration standard peptide, wherein peptide be cut, and it is mixed by enzyme.Can survey the kind specific peptide by the quality fingerprinting analysis, and, for example, use the Mascot search engine to come they are identified by database search, as mentioned below:

Purified natural (n) Der f2 and nDer p2 and reorganization (r) Der f2 (A61501) and Der p2 (BAA01241) can be dissolved in 25mM Tris-Cl, pH 7.5, in the 1.0M urea.The aliquot sample all mixed (for example, 1: 1) of recombinant molecule (rDer f2 and rDer p2) and natural (nDer f2 and nDer p2) molecule, or by trypsinization for dividing other various peptides, mix again afterwards.Can be desalted the digestion product that mixes and/or divide other various peptides, and assess by quality fingerprinting.Can from above-mentioned two kinds potpourri, identify kind of a paraspecific peptide by the quality fingerprinting analysis.After with trypsinization, other HDM 2 allergens of branch through digestion can be mixed the kind specificity constant series that susceptible of proof is same.Can synthetic peptide be designed based on kind specificity constant series.Quantitative and checking to peptide sequence can be used ITRAQ by listing in ^TMReagent to each allergen and calibration standard peptide in addition mark carry out, and analyze by tandem mass spectrum (MS/MS).

Natural allergenic extract, for example, two HDM kinds, dust mite (Dermatophagoides farinae) and dermatophagoides pteronyssinus (Dermatophagoidespteronyssinus) (ALK-Abell ó,

Denmark) intermediate product (1.0mg/ml dry weight) is dissolved.For removing the interference component, available acetone comes deposit sample.Protein-contg precipitation can be dissolved the damping fluid of into selecting for use again then.With regard to quantitative purpose, available, for example, ITRAQ ^TMReagent comes mark to be used as synthetic peptide and sample from two kinds the calibration standard peptide, that select for use, and uses MS/MS that it is analyzed.

With final product, for example, house dust acarid allergen tablet dissolved is in 20mM sodium phosphate buffer (pH 7.0).For removing the interference component, sample may need to be presorted level and separate, and/or uses acetone precipitation.For example use ITRAQ ^TMReagent is to the tablet of dissolving and the calibration standard peptide selected for use mark in addition, and uses series connection MS/MS to analyze.

Releasing research; With final product, for example, 5 careless potpourris comprise with 5 kinds of grass seeds classes of aluminium hydroxide coupling and dissolve the damping fluid of into selecting for use that it can be opened the allergen and the aluminium hydroxide wash-out of combination.Unconjugated allergen can further be separated, and/or the desalination digestion damping fluid that advances to select for use, can for example use ITRAQ ^TMThe calibration standard peptide that reagent comes mark to select for use can use MS/MS to assess quantitatively.

All aspects that any method mentioned in this article and of the present invention is relevant or feature and be equal to relevant undoubtedly according to any other method of the present invention.

Embodiment

Embodiment 1

Identify at the unique constant zone of Der f2, Der p2, Phl p1, Phl p5 and Bet v1 and synthetic calibration standard peptide

Use unique constant regional sequence, that is, the absolute quantitation that the mark peptide among natural Der f2, Der p2, Phl p1, Phl p5 and the Bet v1 carries out different allergen can use two kinds of means of different to show.Synthetic two kinds of calibration standard peptides are used for iTRAQ ^TMLabelling technique (Applied Biosystems) is assessed.In addition, synthetic four kinds of calibration standard peptides are with assessment Protein-AQUA ^TMIsotope labelling techniques such as (Sigma-Aldrich).

Based on amino acid sequence comparison (Vector NTI) (Fig. 1), the cutting analysis (Fig. 2) and the Blast database search that are undertaken by GPMAW, design allergen calibration standard peptide, described standard peptide has the kind specific sequence corresponding to the amino acid sequence of 151-164 among 115-127 and the Bet v1 among 123-135, the Phl p 5b a among 149-158, the Phl p 5a among 32-48, the Phl p1 among Der f2 and the Der p2.To be used to show the existence of kind specific peptide through the Der f2 of natural allergen Der f2, Der p2, Phl p1, Phl p5, Bet v1 and the mixing of trypsinization and external quality fingerprinting analysis that Der p2 carries out.All all are that (MA USA) identifies for Matrix Science Inc., Boston with the Mascot search engine through the sample of external digestion and potpourri.

Can be from Sigma GENOSYS, Texas, US obtains the synthetic peptide corresponding to the 32-48 amino acids sequence of Der f2 and Der p2.(Sigma GENOSYS, Texas US) measure the concentration of these peptides by amino acid analysis.From Sigma GENOSYS, Texas, US obtains corresponding to 149-158 (Arg among the Phl p1 ¹³C ¹⁵N), 123-135 (Arg among the Phl p 5a ¹³C ¹⁵N), 115-127 (Arg among the Phl p 5b ¹³C ¹⁵N) 151-164 (Val and among the Bet v1 ¹³C ¹⁵N) amino acid sequence, stable through isotope-labeled synthetic peptide (Protein-AQUA ^TMPeptide).(table 1)

Embodiment 2

The grass of purifying natural and reorganization, birch and acarid group 2 allergens

(ALK-Abell ó, Horsholm Denmark) come the Der f2 and the Der p2 of purifying natural from 100mg dust mite and dermatophagoides pteronyssinus extract.From the Phl p1 and 5 of 50mg Phleum prantense extract (ALK-Abell ó) purifying natural, come the Bet v1 of purifying natural from 50mg Betulaverrucosa extract (ALK-Abell ó).Purifying to molecule carries out (Johannessen BR et al.FEBS Lett2005 according to document is described; 579:1208-12, Aasmul-Olsen S.et al.New Horizons inAllergy Immunotherapy, edit by Sehon et al.Plenum Press.NewYork 1996, p.261-65, Petersen A et al.Clin Exp Allergy.1994Mar; 24 (3): 250-6, Ipsen H ﹠amp; Lowenstein H J Allergy Clin Immunol.1983; 72 (2): 150-59).Reorganization Der f2 and Der p2 are expressed in the Pichia pastoris expression system, according to document is described it are carried out purifying (Johannessen BR et al.FEBSLett 2005; 579:1208-12).Protein is stored in-20 ℃ as the aliquot sample through freeze-drying.

Use a kind of in the different allergen at A ₂₈₀The extinction coefficient at place, and (Perkin Elmer Instruments, CA USA) measure purified allergenic concentration to use Lambda 800UV/VIS spectrophotometer.

Embodiment 3

Presorting level with the HDM extract separates and protein digestibility

Coming that with hydrophobic interaction chromatograph dust mite (Der f) and dermatophagoides pteronyssinus (Der p) extract are presorted level separates.(GE-Healthcare, Uppsala Sweden) carry out fractionated to the acarid extract with the HiTrap Phenyl post of 1.0ml.With 50mM sodium phosphate buffer (Merck, Darmstadt, Germany), pH 7.0,1.0M ammonium sulfate (Fluka, Buchs, Switzerland) come this post of balance, with the linear gradient that reduces gradually, with the 50mM sodium phosphate buffer (Merck) of 5 times of column volumes, pH 7.0 comes the sample of elution of bound.At in the HDM extract (Der f and Der p) every kind carries out chromatogram respectively.(CA USA) analyzes fraction for Invitrogen, Carlsbad, based on this analysis, HDM albumen branch is gone into two large protein storehouses (Fig. 4) by SDS-PAGE.Carry out 10mM ammonium bicarbonate (BDH, Poole, dialysis England) containing HDM 2 allergenic Der f and Der p storehouse.Der f and Der p storehouse through dialysis are frozen drying, and the aliquot sample that is divided into～5mg (dry weight) tubule of packing into keeps in cold storage in-20 ℃.Contain HDM 2 allergenic Der f and Der p aliquot sample and be further used in absolute quantitation research.

Embodiment 4

Enzymatic cutting and iTRAQ mark

Use iTRAQ from three kinds of sample sets ^TMMark:

A) natural Der f2 of 15 μ g and the natural Der p2 of 15 μ g

B) 15 μ g reorganization Der f2 and 15 μ g reorganization Der p2, and

C) 100 μ g are through presorting Der f and the Der p extract that level is separated.

With synthetic standard peptide, 15 μ g peptides 1 (Der p2) and 15 μ g peptides 2 (Der f2) are dissolved among 100mM triethyl ammonium bicarbonate (TEAB) pH 8.5, and it is carried out mark, to be used as the inside calibration standard at hereinafter described each experimental group.

According to manufacturers protocol, carry out to free cysteine residues sealing, use the enzymatic cutting that trypsase carries out and use iTRAQ ^TMIt is peptide-labeled that reagent carries out:

Six kinds of protein examples and every kind of 100mM TEAB that is dissolved to 20 μ l of two kinds of inner calibration standards, pH 8.5.With the 0.05%SDS of 1.0 μ l to the protein sample sex change, 4.8mM TCEP Tris (2-carboxyethyl) phosphine by 2.0 μ l reduces to it at 60 ℃, then, the 10mM s-methyl first sulphur hydrochlorate (MMTS) by 1.0 μ l is at room temperature sealed cysteine residues.(WI is USA) 37 ℃ of digestion of protein sample being carried out 18 hours for Promega, Madison with the modification trypsase of 10% (w/w) order-checking level.

To sample and the inner calibration standard peptide through trypsinization,

peptide

1 and 2 iTRAQ that carry out ^TMBeing marked at room temperature carries out.Every kind of iTRAQ labelled reagent that will from 114 to 117 is dissolved to 70% ethanol of 70 μ l, itself and then be applied on the sample.The final volume of reagent mixture is 100 μ l/ samples.Sample through mark is stored in-20 ℃.

Peptide with the natural Der p2 of iTRAQ-114 mark, reorganization Der p2 and Der p extract through trypsinization.Peptide with the natural Der f2 of iTRAQ-115 mark, reorganization Der f2 and Derf extract through trypsinization.With iTRAQ-116 (Der p2) and iTRAQ-117 (Der f2) mark synthetic peptide---peptide 1 (Der p2) and peptide 2 (Der f2) are (Fig. 5).Analyze every kind of sample by Voyager STR and/or 4700 Proteomic Analyser (AppliedBiosystems), to show mark to peptide through mark.Carry out the MS/MS fragment analysis with 4700Proteomic Analyser (Applied Biosystems).Sample through mark is carried out dilution in 1: 10, and (ZipTips Millipore) and/or by hand makes C18 micro-column (Poros R2, Applied Biosystems) to 1.0 μ l sample desalinations by C18 Micro post.By with alpha-cyano-4-hydroxyl-cinnamic acid (CHCA) of 1.0 μ l, 70% acetonitriles (ACN), 0.1% trifluoroacetic acid (TFA) and 1.0 μ l (AgilentTechnologies,

Germany) matrix joins the sample top, on target sample is carried out wash-out.Sample is carried out drying, it is carried out MS analyze.

To through iTRAQ ^TMThe MS of the inside calibration standard of mark analyzes the quality corresponding to m/z 2353.44 place's peptides 1, and m/z 2326.35 places are corresponding to the quality (Fig. 6 a and 6b) of peptide 2.The result shows that the modification in Der p2 specific peptide 1 and Der f2 specific peptide 2 is corresponding to the iTRAQ that is attached to the terminal lysine of amino terminal and C ^TMThe modification that reagent carries out.To through iTRAQ ^TMThe MS natural and reorganization Der p2 peptide of mark analyzes and has disclosed m/z 2353.29 places through iTRAQ ^TMThe quality of modifying.Similarly, the MS analysis through the natural of mark and reorganization Der f2 peptide m/z 2326.29 places have been disclosed through iTRAQ ^TMThe quality of modifying is seen Fig. 6 c and 6d respectively.Do not observe the cutting that trypsase is missed.These results show, can be used as inner calibration standard through the

peptide

1 and 2 of iTRAQ mark, are used for Der p2 and the different allergenic absolute quantitation of Der f2 are detected.

Embodiment 5

Absolute quantitation from the potpourri of two kinds of different HDM kinds

The absolute quantitation that different allergen with dust mite and dermatophagoides pteronyssinus kind is carried out is at first from purified natural allergenic direct mixing.Through iTRAQ ^TMMark natural through the Der of trypsin hydrolysis p2, Der f2 and peptide 1 and peptide 2 with 1: 1: 1: 1 ratio is mixed, according to 1: 5 and dilution in 1: 10, by manual system C18 micro-column (Poros R2, AppliedBiosystems) desalination.By using 5 μ g/ μ lCHCA (Sigma) among the 1.0 μ l 70%ACN (Sigma), 0.1%TFA (Fluka) carries out wash-out to sample on target.Carry out the MS/MS fragment analysis by 4700 Proteomic Analyser (Applied Biosystems).

The fragment analysis of m/z 2353.29 has disclosed corresponding at the signals reporter group ion of natural Der p2 and peptide 1, m/z 114 places and m/z 116 places.The different allergenic amount of natural Derp2 is calculated as the ratio of area of the signal area of m/z 114 and m/z 116 (inner calibration standard) (Fig. 7 a) in the sample.The MS/MS fragment analysis of m/z 2326.29 has disclosed corresponding at the signal report ion of natural Der f2 and peptide 2, m/z 115 and m/z 117.

The different allergenic amount of natural Der f2 is calculated as the ratio (Fig. 7 b) of the signal area of m/z 115 and m/z 117 (inner calibration standard) in the sample.Except absolute quantitation, the tabulation of the fragment ions peak of m/z 2353.29 and m/z 2326.29 is submitted to Mascot search engine (MatrixScience) and carries out database analysis.Identify peptide by database analysis as the 32-48 of dust mite and dermatophagoides pteronyssinus HDM allergen 2.

Embodiment 6

By the peptide mixer of two-dimentional chromatographic resolution through mark

Come the potpourri of the different allergen in the complex mixture of Der f and Der p extract and reorganization Der f2 and Der p2 is carried out absolute quantitation by two-dimentional chromatogram.Cation-exchange chromatography (SCX) is used as first dimension, and reverse-phase chromatography is as the second dimension preparation process.

To recombinate Der p2, Der f2 and

peptide

1 and 2 according to 1: 1: 1: 1 ratio is mixed, and final volume is 50 μ l.Der f and Der p extract and

peptide

1 and 2 were according to 4: 4: 1: 1 ratio is mixed, and final volume is 50 μ l.

By cation-exchange chromatography separate peptide mixer through mark (reorganization Der p2 and The extract of Der f2 and Der p2 and Der f2):

According to 1: 10 sample mixture is diluted into 5%ACN (Sigma) 0.05% formic acid (Merck), it is carried out SCX.SCX is at SMART ^TM(GE-HealthCare, Uppsala Sweden) carry out in the Zorbax BIO-SCX of the 0.8 * 50mm in (the 3.5 μ m) post (AgilentTechnologies) in system.Carry out balance with 5%ACN (Sigma) 0.05% formic acid (Merck) coupled columns, use the linear gradient that increases gradually of from 0 to 100% 5%ACN (Sigma) 0.05% formic acid (Merck) 0.5M NaCl (Merck), in 30 minutes, carry out chromatogram.Flow velocity is 50 μ l/ minutes, at the 214nm place chromatogram is monitored.Collect the fraction of 50 μ l, every kind of fraction (1.0 μ l) is analyzed by Voyager-STR MS (Applied Biosystems) and/or 4700 ProteomicAnalyser (Applied Biosystems) equipment.Identify the peptide of m/z 2353.29 and m/z 2326.29 at gradient end wash-out, itself and some other the HDM peptide together.Contain that the Der f of peptide of m/z 2353.29 and m/z 2326.29 and Der p potpourri fraction (extract) are selected comes out, it is further separated, see below by reverse-phase chromatography.But, to quantitatively behind SCX, directly carrying out of inner calibration standard and rDer f2 and rDer p2 (reorganization) potpourri from the target dish.

The MS/MS fragment analysis that m/z 2353.29 is carried out has disclosed corresponding at the signals report ion of Der p2 and peptide 1, m/z 114 and m/z 116 places.Der p2 clone's amount is calculated as the ratio of the area of the signal area of m/z 114 and m/z 116 (inner calibration standard) in the rDer p2/rDer f2 potpourri.The MS/MS fragment analysis that m/z 2326.29 is carried out has disclosed corresponding at the signals reporter group ion of Der f2 and peptide 2, m/z 115 and m/z 117 places.Der f2 clone's amount is calculated as the ratio of the area of the signal area of m/z 115 and m/z 117 (inner calibration standard) in the rDer p2/rDer f2 potpourri.The fragment ions peak tabulation of m/z 2353.29 and m/z 2326.29 is submitted to Mascot search engine (MatrixScience) and carries out database analysis.Identify peptide by database analysis as the 32-48 of Dermatophagoides farinae and Dermatophagoides pteronyssinus HDM allergen 2.

The peptide at m/z 2353.29 and m/z 2326.29 places elutes from the SCX post with close residence time in the potpourri of two duplicate samples---reorganization Der f2 and Der p2 and Der f and the Der p extract mixtures.This tests demonstration, and SCX can be used as fractionated and the desalination step at simpler sample mixture before allergen being carried out quantitatively.Experiment shows that also SCX can be used as the first dimension fractionated step, is used to analyze allergenic more complicated potpourri, the traditional allergenic extract that for example uses in the immunotherapy.

Then carry out reverse-phase chromatography and then carry out absolute quantitation MALDI by SCX TOF-TOF MS separates the peptide (from the extract of Der p2 and Der f2) through mark.

By C18 PepMap100 (3 μ m) post (LC Packings Dionex, Sunny Vale, CA, USA) separate from previously described SCX separate classification through iTRAQ ^TMThe HDM peptide of mark.Come coupled columns to carry out balance with 0.05%TFA (Fluka), 2%ACN (Sigma).With the gradient of 0-50% in 80 minutes, the gradient of 50-100% in 120 minutes is come the wash-out peptide with 0.04%TFA (Fluka), 80%ACN.(LC Packings Dionex) carried out chromatogram in 2.0 μ l/ minutes, 210 and 214nm place monitoring with Ultimate3000.The SCX fraction of 1.0 μ l is injected in the post.By their direct point samples are collected fraction on MALDI-TOF target dish.With Probot (LC Packings, Dionex) equipment comes point sample, this equipment is connected with Ultimate3000 equipment is online.Per 30 seconds according to 1: 1 ratio with HCCA matrix (AgilentTechnologies) and sample mix, carry out point sample.

Use 4700 Proteomic Analyser (Applied Biosystems), analyze the sample of point sample by MS and MS/MS.Point from the target is identified the peptide of m/z 2353.29 and m/z2326.29, and they demonstrate the signal (Fig. 8) corresponding to 214nm place in the chromatogram.The MS/MS fragment analysis that m/z 2353.29 is carried out has disclosed corresponding at the signals reporter group ion of Der p2 and peptide 1, m/z 114 and m/z 116 places.The different allergenic amount of Der p2 is calculated as the ratio of the area of the signal area of m/z 114 and m/z 116 (inner calibration standard) in the Der p/f extract mixtures.The MS/MS fragment analysis that m/z 2326.29 is carried out has disclosed corresponding at the signals reporter group ion of Der f2 and peptide 2, m/z 115 and m/z 117 places.The different allergenic amount of Der f2 is calculated as the ratio of the area of the signal area of m/z 115 and m/z 117 (inner calibration standard) in the Der p/f extract mixtures.The fragment ions peak tabulation of m/z 2353.29 and m/z 2326.29 is submitted to Mascot search engine (MatrixScience) and carries out database analysis.Identify peptide by database analysis as the 32-48 of Dermatophagoides farinae and Dermatophagoides pteronyssinus HDM allergen 2.

This tests demonstration, and reverse-phase chromatography can be used as the second dimension fractionated that the complex mixture to sample and/or allergenic extract carries out, so that different allergen is carried out quantitatively.

Embodiment 7

Use the AQUA strategy that different allergen is carried out absolute quantitation

At AQUA technology (Stemmann O et al.Cell 2001; 107 (6): 715-26, Gerber SA et al.Proc Natl Acad Sci USA 2003; 100 (12): 6940-5.), synthetic mixed stable isotope ( ¹³C, ¹⁵N) peptide is proofreaied and correct in inside, and trypsase carries out the native peptides that enzymatic digestion forms by for example using with simulation.Typically, the molecular mass of mixing peptide through a kind of isotope-labeled amino acid residue changes 6-10Da.With different in the iTRAQ technology, need be modified sample or inner calibration standard by labelled reagent.In quantitative experiment, for example, when using LC-MS/MS, the function that can be used as the reverse-phase chromatography residence time from the abundance of the specific fragment ions of natural sample peptide and synthetic inside calibration standard is measured.By the abundance of known internal standard and the abundance of natural sample peptide are compared, carry out absolute quantitation and measure.

According to the synthetic inner calibration standard peptide (Fig. 2) that designs at natural Phl p1, Phl 1p 5a and b form and Bet v1 mentioned above.Following table 1 has been described synthetic peptide in more detail.

Kind

Allergen

Amino acid sequence

Theoretical Mass

Modify

Modified quality

Betula verrucosa	Bet v1	AVESYLLAHSDAYN	1552.73	(Val ¹³C ¹⁵N)	1558.64
Betula verrucosa	Bet v1	AVESYLLAHSDAYN	1552.73	(Val ¹³C ¹⁵N)	1558.64	Phleum prantense	Phl p1	SAGEVEIQFR	1135.57	(Arg ¹³C ¹⁵N)	1145.27
Phleum prantense	Phl p 5a	YDAYVATLSEALR	1471.74	(Arg ¹³C ¹⁵N)	1481.66	Phleum prantense	Phl p1	SAGEVEIQFR	1135.57	(Arg ¹³C ¹⁵N)	1145.27
Phleum prantense	Phl p 5a	YDAYVATLSEALR	1471.74	(Arg ¹³C ¹⁵N)	1481.66	Phleum prantense	Phl p 5b	FDSFVASLTEALR	1455.74	(Arg ¹³C ¹⁵N)	1465.66

Table 1: to the mark of synthetic inside calibration standard

Natural Phl p1, Phl p5 and Bet v1 are dissolved among 25mM Tris-Cl (Sigma), 1.0M urea (Fluka) pH 7.8 again with the concentration of 2.5pmol/ μ l.Inner calibration standard is mixed into sample with 1: 1 ratio.With the trypsase (Promega) of 10% (w/w) order-checking level 37 ℃ of digestion of carrying out 18 hours.Sample is stored in-20 ℃.

Use Voyager STR and/or 4700 Proteomic Analyser (AppliedBiosystems), analyze through the Phl of trypsinization p1, Phlp5 and Bet v1 by MS and MS/MS.All samples was diluted among the 0.1%TFA (Fluka) according to 1: 10, and every kind of sample is got 1.0 μ l, by handmade C18 micro-column (Poros R2 AppliedBiosystems) to its desalination.

MS through the natural Phl p1 of trypsinization analyzed disclosed the native peptides at m/z 1135.60 places, and m/z 1145.61 places, at the different allergenic inner calibration standard peptide of Phl p1 (data not shown goes out).The MS/MS that inside is proofreaied and correct peptide analyzes the fragmentation pattern that demonstrates corresponding to the amino acid sequence of the different allergenic uniqueness of natural Phl p1.

To analyzing the native peptides of the Phl p 5a that has disclosed m/z 1471.81 places through the MS of the natural Phl p5 of trypsinization, and the native peptides (data not shown goes out) of the Phl P 5b at m/z 1455.77 places.Detected at Phl p 5a and the different allergenic inner calibration standard peptide of Phl p 5b at m/z 1481.83 and m/z 1465.78 places.The MS/MS that inside is proofreaied and correct peptide analyzes the fragmentation pattern that demonstrates corresponding to the amino acid sequence of natural Phl p 5a and the different allergenic uniqueness of Phl p 5b.

Disclosed the native peptides at m/z 1552.76 places to analyzing through the MS of the natural B et of trypsinization v1, and m/z 1558.77 places, at the different allergenic inner calibration standard peptide of Bet v1 (Fig. 9).The MS/MS that inside is proofreaied and correct peptide analyzes the fragmentation pattern that demonstrates corresponding to the amino acid sequence of the different allergenic uniqueness of natural B et v1.

For Betv 1, Phl p1, Phl p 5a and the different allergen of Phl p 5b are carried out absolute quantitation, can be by for example, by the MS/MS equipment of LC coupling, for example LCQ DecaXP (ThermoFinnigan),

Hybrid LC/MS/MS system and 4000 Q TRAPLC/MS/MS systems (Applied Biosystems) analyze sample.

Experiment with the AQUA peptide shows that the synthetic peptide through cold labeling of simulating natural different allergen sequence can be used for the different allergen among natural Phl p1, Phl p5 and the Bet v1 is carried out absolute quantitation.In addition, the analysis that the trypsinization product is carried out does not disclose any cutting that trypsase leaked.SCX can be used for the more complicated allergen potpourri of the inner calibration standard of chemically identical, natural and synthetic property is carried out fractionated with the two-dimentional chromatogram (as described at the iTRAQ chemistry) that RP-HPLC combines.

Claims

1. method is used for the allergenic absolute magnitude of allergen sample quantitatively in addition, and described method comprises the steps:

A) provide one or more allergen calibration standard peptides of known quantity, described standard peptide have with treat quantitative allergen in the identical amino acid sequence of sequence found, and alternatively, to described allergen calibration standard peptide mark in addition,

B) the described allergen sample of degraded, obtaining the potpourri of peptide, and alternatively, with one or more labelled reagents to described peptide mark in addition,

Wherein, have at least equally in peptide in the described allergen sample of degraded or the described calibration standard peptide and be labeled, if the two all has been labeled peptide in the allergen sample of described degraded and described allergen calibration standard peptide, the described labelled reagent that is used for the described allergen calibration standard of mark peptide is different with the described labelled reagent of the peptide of the allergen sample that is used for the described degraded of mark

C) by quality analysis, the amount of the corresponding peptide in the allergen sample of the amount of described allergen calibration standard peptide and described degraded is associated, come allergenic described absolute magnitude quantitative in addition.

2. the method for claim 1 is used for the allergenic absolute magnitude of allergen sample quantitatively in addition, and described method comprises the steps:

A) use with step b) in the used different labelled reagent of labelled reagent, come one or more allergen calibration standard peptides of mark known quantity, described standard peptide have with treat quantitative allergen in the identical amino acid sequence of sequence found,

B) the described allergen sample of degraded, obtaining the potpourri of peptide, and with one or more labelled reagents to the peptide that obtained mark in addition,

C) by quality analysis, the amount of the corresponding peptide in the allergen sample of the amount of described allergen calibration standard peptide through mark and described degraded is associated, come allergenic described absolute magnitude quantitative in addition.

3. the method for claim 1 is used for the allergenic absolute magnitude of allergen sample quantitatively in addition, and described method comprises the steps:

A) come one or more allergen calibration standard peptides of mark known quantity with labelled reagent, described standard peptide have with treat quantitative allergen in the identical amino acid sequence of sequence found,

B) the described allergen sample of degraded, with the potpourri of acquisition peptide,

4. the method for claim 1 is used for the allergenic absolute magnitude of allergen sample quantitatively in addition, and described method comprises the steps:

A) provide one or more allergen calibration standard peptides of known quantity, described standard peptide have with treat quantitative allergen in the identical amino acid sequence of sequence found,

C) by quality analysis, with described in the allergen sample of the amount of described allergen calibration standard peptide and described degraded accordingly the amount through the peptide of mark associate, come allergenic described absolute magnitude in addition quantitative.

5. as any described method among the claim 1-4, wherein, described allergen calibration standard peptide have with the peptide that obtains by degraded according to step b) in the identical amino acid sequence of amino acid sequence.

6. as the described method of claim 1-5, wherein, treat that quantitative allergen is by constituting more than a kind of different allergen.

7. as the described method of claim 1-5, wherein, treat that quantitative allergen is by constituting more than a kind of homology allergen.

8. as any described method among the above-mentioned claim 1-7, wherein, the described quantitative use mass spectrum in the step c) carries out.

9. as any described method among the above-mentioned claim 1-8, wherein, treat that quantitative allergen is to be selected from the p1 by Phl, Phl p5, Phl p6, Poa p1, Poa p5, Dac g1, Fes p1, Lol p1, Lol p5, Der f1, Der f2, Der p1, Der p2, Api m1, Api m2, Ves v1, Ves v2, Ves v5, Dolm1, Dol m2, Dol m5, Dol a5, Pol a1, Pol a2, Pol a5, Amb a1, Amb a2, Par j1, Par o1, Par m1, Bet v1, Cry j1, Cry j2, Per a1, Ole e1, Fel d1, Can f1, Can f2, Equ c1, Equ c2, Art v1, Art v2, Art v3, Alt a1, Alt a3, Alt a4, Alt a5, Alt a6, Cla h1, Cla h2, Cla h6, Sol i2, one or more different allergens in the group that Soli3 and Sol i4 constitute.

10. method 9 as claimed in claim, wherein, treat that quantitative allergen is one or more different allergens that are selected from the group that is made of Phl p1, Phl p5, Phl p6, Ole e1, Der f1, Der f2, Der p1, Der p2, Ves v1, Ves v2, Ves v5, Amb a1, Amb a2, Parj1, Par o1, Par m1, Bet v1, Cry j1 and Cry j2.

11. method as claimed in claim 10 wherein, treats that quantitative allergen is one or more different allergens that are selected from the group that is made of Der f1, Der p1, Der f2 and Der p2.

12. method as claimed in claim 9 wherein, treats that quantitative allergen is one or more different allergens that are selected from the group that is made of Phl p1, Phl p5, Phl p6, Poa p1, Poa p5, Dac g1, Fes p1, Lol p1 and Lol p5.

13. method as claimed in claim 9 wherein, treats that quantitative allergen is one or more different allergens that are selected from the group that is made of Amb a1 and Amb a2.

14. any as described above described method of claim, wherein, described mark is to use ITRAQ ^TMChemistry carries out.

15. method as claimed in claim 14, wherein, described mark carries out with ITRAQ-114, ITRAQ 115, ITRAQ 116 and/or ITRAQ 117.

16. any as described above described method of claim, wherein, (i) treat that quantitative described allergen is Der f2, described allergen standard correction peptide comprises the 32-48 amino acids of Der f2, or treat that (ii) quantitative described allergen is Der p2, described allergen standard correction peptide comprises the 32-48 amino acids of Der p2.

17. any as described above described method of claim wherein, by the peptide identification and analysis, is compared described allergen peptide mixer and allergen calibration standard peptide, clearly identifies described allergen.

18. any as described above described method of claim, wherein, by digest the described allergen sample of degrading with at least a enzyme, with the described sample of part or all of degraded.

19. method as claimed in claim 18, wherein, described enzyme is a proteolytic enzyme.

20. method as claimed in claim 19, wherein, described proteolytic enzyme is selected from trypsase, papain, pepsin, ArgC, LysC, V8 proteinase, AspN, pronase, chymotrypsin and carboxypeptidase C or its group that constitutes.

21. method as claimed in claim 20, wherein, described enzyme is selected from the group that ArgC, LysC and trypsase or its constitute.

22. method as claimed in claim 21, wherein, described enzyme is a trypsase.

23. any as described above described method of claim wherein, before degraded, is reduced to described allergen sample, and is sealed any cysteine residues.

24. any as described above described method of claim, wherein, described allergen sample is to separate the fraction of step from presorting level.

25. method as claimed in claim 24, wherein, described allergen sample is to separate the fraction of step from presorting level, and it comprises described allergenic one or more different allergens.

26. as the described method of claim 24-25, wherein, the described level of presorting is separated and to be undertaken by chromatogram.

27. method as claimed in claim 26, wherein, described chromatogram fractionated is undertaken by hydrophobic interaction chromatograph, reverse-phase chromatography, ion-exchange chromatography, size exclusion chromatogram or affinity chromatography.

28. method as claimed in claim 27, wherein, described chromatogram fractionated is undertaken by hydrophobic interaction chromatograph.

29. as the described method of claim 24-28, wherein, described allergen sample experiences desalination after chromatogram.

30. any as described above described method of claim, wherein, before quality analysis, to described degraded and carry out fractionated through the peptide mixer and the described calibration standard peptide of mark alternatively, separating corresponding to the peptide of described calibration standard peptide and other peptide in the described potpourri.

31. method as claimed in claim 30, wherein, described fractionated is undertaken by chromatogram.

32. method as claimed in claim 31, wherein, described fractionated is undertaken by then carrying out reverse-phase chromatography after cation-exchange chromatography or the cation-exchange chromatography.

33. a method is used for specific allergen or different allergen from the allergen sample quantitatively in addition, described method comprises:

A) provide one or more allergen calibration standard peptides of known quantity, it has unique amino acid sequence the sequence of finding in treating quantitative described allergen or different allergen, and, alternatively, to described allergen calibration standard peptide mark in addition,

Wherein, have at least equally in peptide in the described degraded sample or the described calibration standard peptide and be labeled, if the two all has been labeled peptide in the described degraded sample and described allergen calibration standard peptide, the labelled reagent that is used for the described allergen calibration standard of mark peptide is different with the labelled reagent of the peptide that is used for the described degraded sample of mark

C) by quality analysis, the amount of described allergen calibration standard peptide and the amount of the corresponding peptide in the described degraded sample are associated, come allergenic described absolute magnitude quantitative in addition.

34. method as claimed in claim 33 is used for the allergenic absolute magnitude of allergen sample quantitatively in addition, described method comprises the steps:

A) use with step b) in the used different labelled reagent of labelled reagent, come one or more allergen calibration standard peptides of mark known quantity, described standard peptide has amino acid sequence unique the sequence of finding in treating quantitative described allergen or different allergen

C) by quality analysis, the amount of described allergen calibration standard peptide through mark and the amount of the corresponding peptide in the described degraded sample are associated, come allergenic described absolute magnitude quantitative in addition.

35. method as claimed in claim 33 is used for the allergenic absolute magnitude of allergen sample quantitatively in addition, described method comprises the steps:

A) come one or more allergen calibration standard peptides of mark known quantity with labelled reagent, described standard peptide has unique amino acid sequence the sequence of finding in treating quantitative described allergen,

36. method as claimed in claim 33 is used for the allergenic absolute magnitude of allergen sample quantitatively in addition, described method comprises the steps:

A) provide one or more allergen calibration standard peptides of known quantity, described standard peptide has unique amino acid sequence the sequence of finding in treating quantitative described allergen,

C),, come allergenic described absolute magnitude in addition quantitative with associating through the amount of the peptide of mark accordingly in the amount of described allergen calibration standard peptide and the described degraded sample by quality analysis.

37. as any described method among the claim 33-36, wherein, described allergen calibration standard peptide have with by the identical amino acid sequence of amino acid sequence in the peptide that obtains according to the degraded of step b).

38. as the described method of claim 33-37, wherein, described allergen is different allergen.

39. as the described method of claim 33-38, wherein, described allergen is to be selected from Phlp1, Phl p5, Phl p6, Poa p1, Poa p5, Dac g1, Fes p1, Lol p1, Lol p5, Der f1, Der f2, Der p1, Der p2, Api m1, Api m2, Ves v1, Ves v2, Ves v5, Dol m1, Dol m2, Dolm5, Dol a5, Pol a1, Pol a2, Pol a5,, Amb a1, Amb a2, Parj1, Par o1, Par m1, Bet v1, Cry j1, Cry j2, Per a1, Ole e1, Fel d1, Can f1, Can f2, Equ c1, Equ c2, Art v1, Art v2, Art v3, Alt a1, Alt a3, Alt a4, Alt a5, Alta6, Cla h1, Cla h2, Cla h6, Sol i2, the allergenic different allergen of the group that Sol i3 and Sol i4 constitute.

40. method as claimed in claim 39, wherein, described allergen is the allergenic different allergen that is selected from the group of Phl p1, Phl p5, Phl p6, Ole e1, Der f1, Der f2, Der p1, Derp2, Ves v1, Ves v2, Ves v5, Amb a1, Amb a2, Par j1, Par o1, Par m1, Bet v1, Cry j1 and Cry j2 formation.

41. as any described method among the claim 33-40, wherein, quantitatively carrying out in the step c) with mass spectrum.

42. as any described method among the claim 33-41, wherein, described mark is to use ITRAQ ^TMChemistry carries out.

43. as any described method among the claim 33-42, wherein,, described allergen peptide mixer and allergen calibration standard peptide through mark compared, clearly identified described different allergen by the peptide identification and analysis.

44. as any described method among the claim 33-43, wherein, by digest the described allergen sample of degrading with at least a enzyme, with the described sample of part or all of degraded.

45. method as claimed in claim 44, wherein, described enzyme is a proteolytic enzyme.

46. method as claimed in claim 45, wherein, described proteolytic enzyme is selected from trypsase, papain, pepsin, ArgC, LysC, V8 proteinase, AspN, pronase, chymotrypsin and carboxypeptidase C or its group that constitutes.

47. method as claimed in claim 46, wherein, described enzyme is selected from the group that ArgC, LysC and trypsase or its constitute.

48. method as claimed in claim 47, wherein, described enzyme is a trypsase.

49., wherein, before degraded, described allergen sample is reduced, and seals any cysteine residues as any described method among the claim 33-48.

50. as any described method among the claim 33-49, wherein, described allergen sample is to separate the fraction of step from presorting level.

51. method as claimed in claim 50, wherein, described allergen sample is to separate the fraction of step from presorting level, and it comprises different allergen.

52. as any described method among the claim 50-51, wherein, the described level of presorting is separated and to be undertaken by chromatogram.

53. method as claimed in claim 52, wherein, described chromatogram fractionated is undertaken by hydrophobic interaction chromatograph, reverse-phase chromatography, ion-exchange chromatography, size exclusion chromatogram or affinity chromatography.

54. method as claimed in claim 53, wherein, described chromatogram fractionated is undertaken by hydrophobic interaction chromatograph.

55. as any described method among the claim 33-54, wherein, described allergen sample experiences desalination after chromatogram.

56. as the described method of claim 33-52, wherein, before quality analysis, to described degraded and carry out fractionated through the peptide mixer and the described calibration standard peptide of mark alternatively, separating corresponding to the peptide of described calibration standard peptide and other peptide in the described potpourri.

57. method as claimed in claim 56, wherein, described fractionated is undertaken by chromatogram.

58. method as claimed in claim 57, wherein, described fractionated is undertaken by then carrying out reverse-phase chromatography after cation-exchange chromatography or the cation-exchange chromatography.

59. as above-mentioned any described method of claim, wherein, amino acid whose quantity is in 2-20 amino acid whose scope, more preferably in the scope of 4-15, most preferably in the scope of 6-10 in the allergen calibration standard peptide.

60. a method is used to obtain to be used for allergen is carried out quantitative allergen calibration standard peptide, wherein, described allergen calibration standard peptide is to obtain like this:

By more different allergen or the allergenic amino acid sequence of homology, identify the constant amino acid sequence for the treatment of in the quantitative described allergen, and preparation has the synthetic property allergen calibration standard peptide of this constant series.

61. method 60 as claimed in claim, wherein, described allergen is allergenic different allergen.

62. following amino acid sequence is used for allergen is carried out absolute quantitation as allergen calibration standard peptide, and the purposes of alternatively allergen being identified, wherein, the sequence of described amino acid sequence with treat quantitative allergen in the sequence found identical.

63. following amino acid sequence is used for allergen is carried out absolute quantitation as allergen calibration standard peptide, and the purposes of alternatively allergen being identified, wherein, the sequence of described amino acid sequence with treat quantitative allergenic different allergen in the sequence found identical.

64. a method is used to obtain to be used for allergen is carried out quantitative allergen calibration standard peptide, wherein, described allergen calibration standard peptide is to obtain like this:

By more different allergen or the allergenic amino acid sequence of homology, identify amino acid sequence unique for treating quantitative described allergen, and prepare synthetic property allergen calibration standard peptide with this sequence.

65. as the described method of claim 64, wherein, described allergen is different allergen.

66. following amino acid sequence is used for allergen is carried out absolute quantitation as allergen calibration standard peptide, and the purposes of alternatively allergen being identified, wherein, the sequence of described amino acid sequence is unique for treating quantitative allergen.

67. following amino acid sequence is used for different allergen is carried out absolute quantitation as allergen calibration standard peptide, and alternatively to different should the former purposes of identifying, wherein, the sequence of described amino acid sequence is unique should be former for treating quantitative mutation.